Methods and kits for synthesis of siRNA expression cassettes

ABSTRACT

Amplification-based methods and kits for rapidly producing siRNA expression cassettes are provided. Also provided are methods for expressing amplified siRNA expression cassettes in cells.

This application claims priority to U.S. Provisional Application Ser.Nos. 60/399,718 filed Aug. 1, 2002 and 60/408,298 filed Sep. 6, 2002.

GOVERNMENT RIGHTS STATEMENT

This invention was made with federal government support from theNational Institutes of Health of the U.S. Department of Health and HumanServices under Grant No. AI29329 to the City of Hope Cancer Center. TheUnited States government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to RNA interference (RNAi), and is usefulfor screening multiple RNAi gene constructs to identify those mosteffective against a given target.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is a process in which double stranded RNA (dsRNA) induces the postranscriptional degradation of homologoustranscripts, and has been observed in a variety of organisms includingplants, fungi, insects, protozans, and mammals. (Moss, E. G., et al.,2001; Bernstein, E., et al., 2001; Elbashir, S. M., et al., 2001;Elbashir, S. M., et al., 2001). RNAi is initiated by exposing cells todsRNA either via transfection or endogenous expression. Double-strandedRNAs are processed into 21 to 23 nucleotide (nt) fragments known assiRNA (small interfering RNAs). (Elbashir, S. M., et al., 2001;Elbashir, S. M., et al., 2001). These siRNAs form a complex known as theRNA Induced Silencing Complex or RISC (Bernstein, E., et al., Hammond,S. M., et al. 2001), which functions in homologous target RNAdestruction. In mammalian systems, the sequence specific RNAi effect canbe observed by introduction of siRNAs either via transfection orendogenous expression of 21-23 base transcripts or longer hairpinprecursors. Use of siRNAs evades the dsRNA induced interferon and PKRpathways that lead to non-specific inhibition of gene expression.(Elbashir, S. M., et al., 2001).

Recently, several groups have demonstrated that siRNAs can beeffectively transcribed by Pol III promoters in human cells and elicittarget specific mRNA degradation. (Lee, N. S., et al., 2002; Miyagishi,M., et al., 2002; Paul, C. P., et al., 2002; Brummelkamp, T. R., et al.,2002; Ketting, R. F., et al., 2001). These siRNA encoding genes havebeen transiently transfected into human cells using plasmid or episomalviral backbones for delivery. Transient siRNA expression can be usefulfor rapid phenotypic determinations preliminary to making constructsdesigned to obtain long term siRNA expression. Of particular interest isthe fact that not all sites along a given mRNA are equally sensitive tosiRNA mediated downregulation. (Elbashir, S. M., et al., 2001; Lee, N.S., et al., 2001; Yu, J. Y., et al., 2002; Holen, T, et al., 2002).

In contrast to post-transcriptional silencing involving degradation ofmRNA by short siRNAs, the use of long siRNAs to methylate DNA has beenshown to provide an alternate means of gene silencing in plants.(Hamilton, et al.). In higher order eukaryotes, DNA is methylated atcytosines located 5′ to guanosine in the CpG dinucleotide. Thismodification has important regulatory effects on gene expression,especially when involving CpG-rich areas known as CpG islands, locatedin the promoter regions of many genes. While almost all gene-associatedislands are protected from methylation on autosomal chromosomes,extensive methylation of CpG islands has been associated withtranscriptional inactivation of selected imprinted genes and genes onthe inactive X-chromosomes of females. Aberrant methylation of normallyunmethylated CpG islands has been documented as a relatively frequentevent in immortalized and transformed cells and has been associated withtranscriptional inactivation of defined tumor suppressor genes in humancancers. In this last situation, promoter region hypermethylation standsas an alternative to coding region mutations in eliminating tumorsuppression gene function. (Herman, et al.). The use of siRNAs fordirecting methylation of a target gene is described in U.S. ProvisionalApplication No. 60/447,013, filed Feb. 13, 2003, which is incorporatedherein by reference.

There are at this time no rules governing siRNA target site selectionfor a given mRNA target. It is therefore important to be able to rapidlyscreen potential target sequences to identify a sequence or sequencessusceptible to siRNA mediated degradation. Initial attempts ataddressing this problem have taken advantage of anoligonucleotide/RNAseH procedure in cell extracts on native mRNAtranscripts designed to identify sites that are accessible tobase-paring, including pairing by nucleic acid products such asribozymes. This approach has also been applied to identifying bindingsites for siRNA (Lee, N. S. et al. 2001). Having identified anaccessible site with the oligonucleotide/RNAseH procedure it is stillnecessary to generate siRNAs against the target at the accessible site.This approach has been applied to siRNA site accessibility as well.(Lee, N. S., et al., 2001). However, this process can be time consuming,and in the end it is still necessary to synthesize the siRNA genes forfinal testing.

Thus, an object of the present invention is to provide anamplification-based approach in the form of a method and kit for rapidlysynthesizing siRNA genes, so as to permit rapid screening of potentialtarget sequences susceptible to siRNA mediated degradation.

Another object of the invention is to provide a method for controllingor inhibiting expression of a target gene by transfecting a cell with anamplified siRNA expression cassette.

SUMMARY OF THE INVENTION

The present invention provides an amplification-based approach (e.g.,Polymerase Chain Reaction (PCR)) for rapid synthesis ofpromoter-containing siRNA expression cassettes, and their subsequenttransfection into cells. This approach, which includes methods and kitsfor performing the methods, can be utilized for the facile screening ofsiRNA encoding genes to identify those encoding siRNAs having the bestfunctional activity for a given target. The approach can be utilizedwith siRNAs expressed independently from promoters or with siRNAsexpressed as hairpin precursors or other precursors. The amplificationproducts produced using the approach may be used directly, withoutsubsequent cloning, by transfecting them into cells followed byfunctional assays.

The method of the present invention is fast and inexpensive, allowingmultiple different siRNA gene candidates and/or promoter candidates tobe rapidly screened for efficacy before cloning into a vector.

The method of the present invention is useful for screening siRNA genelibraries.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a PCR strategy used to yield U6transcription cassettes expressing siRNAs. The 5′ PCR primer iscomplementary to the 5′ end of the U6 promoter and is standard for allPCR reactions. A) The 3′ PCR primer is complementary to sequences at the3′ end of the U6 promoter and is followed by either the sense orantisense sequences, a stretch of four to six deoxyadenosines (Ter) andan additional “stuffer-Tag” sequence. The adenosines are the terminationsignal for the U6 Pol III promoter; therefore, any sequence added afterthis signal will not be transcribed by the Pol III polymerase and willnot be part of the siRNA. B) The sense and antisense sequences arelinked by a 9 nt loop and are inserted in the cassette by a two-step PCRreaction. C) The sense and antisense sequences linked by a 9-nucleotideloop and followed by the stretch of adenosines and by the Tag sequencesare included in a single 3′ primer. D) Complete PCR expression cassetteobtained by the PCR reaction. To amplify and identify functional siRNAsfrom the transfected cells, or to increase the yield of the PCR productshown in D, a nested PCR can be performed using the universal 5′ U6primer and a 3′ primer complementary to the Tag sequence (alsostandard), as indicated in the figure.

FIG. 2 shows the inhibition of enhanced green fluorescent protein (EGFP)expression using siRNA-containing PCR cassettes transfected in 293cells. The PCR cassettes containing either the sense, antisense, or bothsense and antisense siRNAs were co-transfected with the target constructinto 293 cells expressing the Ecdysone trans-activator. The humanimmunodeficiency virus (HIV) rev target is fused to the greenfluorescent protein mRNA which is expressed from an inducible promoter.After adding Ponasterone A, EGFP expression can be detected in thecontrol cells (A), but not in cells transfected with either a mixture ofsense and antisense siRNA expressing PCR products (D), or with the PCRcassette expressing the hairpin construct (E). Panels B and C depictco-transfection of cells with target and PCR cassettes expressing sensealone (B) or antisense alone (C). The Rev-EGFP protein is primarily inthe cell nucleolus as a consequence of the nucleolar localizing signalin the Rev portion.

FIG. 3 shows the detection of siRNAs and PCR amplification of siRNAencoding DNAs in transfected cells. A. Northern gel analyses of siRNAsexpressed from PCR products transfected in A293 cells. Lane 1, cellstransfected with the EFGP target construct alone; Lane 2, cellstransfected with antisense encoding construct alone; Lane 3, cellsco-transfected with antisense and sense encoding constructs; Lane 4,cells transfected with hairpin expression construct. The probe iscomplementary to the antisense. In Lanes 24, the siRNA encoding DNAconstructs were co-transfected with the inducible EGFP construct. Thehairpin product appears smaller than the individually expressed siRNAs,demonstrating processing of the hairpin loop. B and C. PCR amplificationof transfected PCR constructs. B. PCR amplification of non-specificsiRNA encoding DNA from fluorescence activated cell sorting (FACS)sorted EFGP positive and negative cells. The non-functional construct isdetected in all cell fractions. Lanes 1 and 4 show the amplificationresults from the EGFP positive fractions. Lanes 2 and 3 show theamplification results from the EGFP negative fractions. C. PCRamplification of functional hairpin expression construct from FACSsorted, EGFP expressing and non-expressing cells. The amplificationresults show the presence of the functional siRNA only in the EGFPnegative fractions (lanes 2-3). In lane 4, there is a small amount ofamplified product, perhaps derived from some contaminating of EGFPnegative cells. NC indicates negative PCR controls.

FIG. 4 is a graph showing a comparison of HIV inhibition by shRNAsexpressed from a PCR product and plasmids.

FIG. 5 is a graph showing the persistence of HIV inhibition by shRNAsexpressed from PCR products in accordance with the present invention.

FIG. 6 is a graph showing the results of an HIV inhibition test usingcloned U6+1 shRNA constructs.

FIG. 7 is a graph showing the results of HIV inhibition assays using apurified U6+1 shRNA PCR product in accordance with the presentinvention.

FIG. 8 is a graph showing the results of HIV inhibition assays using apurified U6+1 shRNA product in accordance with the present invention.

FIG. 9 shows schematically an embodiment of the present invention inwhich a PCR-amplified siRNA expression cassette is cloned into a cloningvector.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an amplification-based method forproducing a promoter-containing siRNA expression cassette.

In one embodiment, the method comprises:

-   -   i) treating one strand of a double-stranded promoter sequence or        construct, in an amplification reaction mixture, with an        oligonucleotide primer which is complementary to the 5′ end of        the promoter sequence;    -   ii) treating the other strand of the promoter sequence or        construct, in the amplification reaction mixture, with a second        oligonucleotide primer which is complementary to the 3′ end of        the promoter sequence, wherein the second primer comprises one        or more sequences which are complementary to a sequence encoding        a sense or (and/or) antisense sequence of a siRNA molecule,        optionally along with one or both of a loop sequence and a        terminator sequence; and    -   iii) treating the amplification reaction mixture of steps (i)        and (ii) in an amplification reaction at a temperature for        annealing and extending said primers on the promoter sequence or        construct and at a temperature for denaturing the extension        products to provide an amplified product comprising the        promoter, one or more sequences encoding the sense or (and/or)        antisense sequence of the siRNA, and one or both of the loop        sequence and the terminator sequence.

The steps (i)-(iii) can be repeated a sufficient number of times toamplify and detect the promoter-containing siRNA expression cassette. Itis also recognized that alternatives to the loop sequence and/orterminator sequence may be utilized in the invention, which are capableof achieving the same function or purpose as the loop and terminatorsequences. It is also recognized that variations in the above steps areencompassed within the invention, in the event these variations alsoprovide an amplification-based method for producing apromoter-containing siRNA expression cassette. It is further recognizedthat the term complementary, although in a preferred embodiment refersto a perfect base-paired match between two sequences, may not requiresuch, and thus the term complementary also encompasses those sequencesnot having a perfect base-paired match but which are otherwise able toachieve the intended result of the invention.

The terms “loop sequence” and “terminator sequence” refer to thesequences corresponding to the loop and terminator elements, includingthe final sequences and any precursor sequences such as the sequencesencoding the final sequences, and any complementary sequences.

In a preferred embodiment, the method is a PCR-based method. However, itis recognized that the invention may be practiced based on otheramplification methods known currently or in the future.

The promoter may be any promoter capable of transcribing an siRNAmolecule, and is preferably one that can transcribe siRNA in mammaliancells. In a preferred embodiment, the promoter is a Pol III promoter,more preferably a mammalian U6 promoter, and most preferably a human U6promoter. Other promoters, such as the H1 promoter, U1 or tRNA promoterssuch as tRNA Val, Met or Lys3 may also be useful in the presentinvention. It is also possible to use Pol II promoters such as the U1snRNA promoter.

The terminator sequence may be any sequence encoding a functionalterminator sequence. In a preferred embodiment, the terminator sequencecomprises a sequence of deoxyadenosines, preferably about 4-6deoxyadenosines, and more preferably a sequence of 6 deoxyadenosines.

In another embodiment, the second primer may further comprise a tagsequence to identify functional siRNA encoding sequences. In a morepreferred embodiment, the tag sequence may further comprise arestriction site useful for cloning.

In one embodiment, the second primer comprises a sequence that iscomplementary to a sequence encoding a sense sequence, along with aterminator sequence or loop sequence.

In another embodiment, the second primer comprises a sequence that iscomplementary to a sequence encoding an antisense sequence, along with aterminator sequence or loop sequence.

In still another embodiment, the second primer comprises a sequence thatis complementary to a sequence encoding a sense sequence and a sequencethat is complementary to a sequence encoding an antisense sequence ofsaid siRNA molecule, along with a terminator sequence.

In a preferred embodiment of the above embodiment, the sense andantisense sequences may be attached by a loop sequence. The loopsequence may comprise any sequence or length that allows expression of afunctional siRNA expression cassette in accordance with the invention.In a preferred embodiment, the loop sequence contains higher amounts ofuridines and guanines than other nucleotide bases. The preferred lengthof the loop sequence is about 4 to about 9 nucleotide bases, and mostpreferably about 8 or 9 nucleotide bases.

The amplified products of the present method will vary depending onwhich embodiment above is selected.

The sequences or constructs encoding the sense and antisense sequencespreferably contain about 19-29 nucleotides, more preferably about 19-23nucleotides, and most preferably about 21 nucleotides. The siRNAmolecules also may contain 3′ nucleotide, preferably 3′ dinucleotideoverhangs, including 3′UU. More generally, the RNAi or siRNA moleculesalso include those known in the art.

In one embodiment, the amplified product comprises the promoter and asequence or construct encoding either the sense or antisense sequence ofthe siRNA molecule. The amplified product also may contain the loopsequence or the terminator sequence.

In another embodiment, the amplified product comprises the promoter, asequence or construct encoding either the sense or antisense sequence ofthe siRNA molecule, and the terminator sequence.

In another embodiment, the amplified product comprises the promoter, asequence or construct encoding either the sense or antisense sequence ofthe siRNA molecule, and the loop sequence. In this embodiment, theamplified product may be treated in another amplification reaction toprovide another amplified product. This may be achieved using a thirdoligonucleotide primer. A portion of this third primer is complementaryto the loop sequence of the first amplified product. The third primeralso comprises a sequence complementary to a sequence encoding theantisense sequence when the first amplified product contains the senseencoding sequence, or a sequence complementary to a sequence encodingthe sense sequence when the first amplified product contains theantisense encoding sequence. The third primer also may include aterminator sequence.

In another embodiment, the amplified product comprises the promoter, asequence or construct encoding the sense sequence and a sequence orconstruct encoding the antisense sequence of the siRNA molecule. In thisembodiment, the sense and antisense encoding sequences or constructs maybe attached by a loop sequence. The amplified product also may contain aterminator sequence.

In still another embodiment, amplified products are produced thatcomprise the promoter, a sequence or construct encoding the sensesequence and a sequence or construct encoding the antisense sequence ofthe siRNA molecule. The sense and antisense encoding sequences orconstructs may be attached by a loop sequence. The amplified productsalso may contain a terminator sequence.

In yet another embodiment, the amplified product, in anotheramplification reaction, can be treated with a fourth oligonucleotideprimer, a portion of which is complementary with the tag sequence.

In a preferred embodiment, the method may further comprise the step ofpurifying the amplified promoter-containing siRNA expression cassette.Various purification techniques are known in the art and may be used inthe present invention. Examples are described below.

In another embodiment, the amplified, and preferably purified,promoter-containing siRNA expression cassette produced according to theinvention is transfected into cells for screening. After transfection,the siRNA can be expressed to induce gene silencing.

In another embodiment, a selected and preferably purified,promoter-containing siRNA expression cassette is cloned into a selectedvector. For this embodiment, it is recognized that restriction sites canbe inserted at the ends of the siRNA expression cassette, preferablyduring production, for example, by including restriction site-encodingsequences within the primers. A schematic of this embodiment is shown inFIG. 9, as well as in U.S. Provisional Application No. 60/399,397, filedJul. 31, 2002, which is incorporated herein by reference.

In a preferred embodiment, the selected cells are mammalian cells.

In another preferred embodiment, one or more of the oligonucleotideprimers are modified, preferably by phosphorylation.

In another embodiment, the method also comprises the step of screeningfor a target site on mRNA sensitive to the expressed siRNA molecule.

In another embodiment, the method includes a positive and/or negativecontrol, such as a control cassette.

In another aspect, the invention provides a method for inhibitingexpression of a target gene. The method comprises transfecting a cellwith an amplified, and preferably purified, siRNA expression cassette sothat a siRNA can be expressed and inhibit the target gene. In apreferred embodiment, the cell is transfected with two or more differentsiRNA expression cassettes. In one embodiment, the different siRNAexpression cassettes contain different siRNA encoding genes, includingdifferent loop sequences, and/or different promoters.

In another aspect, the invention provides a method for modifying genefunction in mammals, for example by directing methylation of a targetgene, including a promoter region of the gene, by transfecting a cellwith an amplified siRNA expression cassette in accordance with theinvention.

In another aspect, the invention provides a PCR-based approach in theform of a kit for producing a promoter-containing siRNA expressioncassette. The kit comprises a double-stranded, promoter-containingtemplate, an oligonucleotide primer complementary to the 5′ end of thepromoter-containing template, and an oligonucleotide primercomplementary to the 3′ end of the promoter-containing template. The 3′primer also comprises one or more sequences complementary to a sequenceencoding a sense or (and/or) antisense sequence of a siRNA molecule.

The 3′ primer may further comprise a loop sequence, in which case thekit further comprises an oligonucleotide primer complementary to theloop sequence, which primer comprises a sequence complementary to asequence encoding a sense or antisense sequence of the siRNA molecule.

In one embodiment, the kit comprises a 3′ primer comprising a sequencecomplementary to a sequence encoding a sense sequence and another 3′primer comprising a sequence complementary to a sequence encoding anantisense sequence.

In another embodiment, the 3′ primer comprises a sequence that iscomplementary to a sequence encoding a sense sequence, a sequence thatis complementary to a sequence encoding an antisense sequence, and aterminator sequence. The sequences complementary to the sense andantisense encoding sequences preferably are attached by a loop sequence.

In a preferred embodiment, the oligonucleotide primers are modified,preferably by phosphorylation.

The kit also may comprise PCR amplification reagents and reagents forpurifying the amplified siRNA expression cassette.

In another preferred embodiment, the kit also comprises one or both of apositive and negative control.

Preferred embodiments of the invention are described below; however, theinvention is understood not to be limited to the following embodiments.

PCR Amplification, Transfection, and Expression of siRNAs in MammalianCells.

The procedure for a PCR-based approach is depicted schematically inFIG. 1. In a preferred embodiment, universal primer that iscomplementary to the 5′ end of the human U6 promoter is used in a PCRreaction along with a primer(s) complementary to the 3′ end of thepromoter, which primer harbors appended sequences which arecomplementary to the sense or antisense siRNA genes (FIG. 1A). The senseor antisense sequences are followed by a transcription terminatorsequence (Ter), which is preferably a stretch of about 4-6deoxyadenosines, and more preferably a stretch of 6 deoxyadenosines, andby a short additional “stuffer-tag” sequence that may include arestriction site for possible cloning at a later stage. The resultingPCR products include the U6 promoter sequence, the siRNA sense orantisense encoding sequence, a terminator sequence, and a short tagsequence at the 3′ terminus of the product.

In another embodiment, both the sense and antisense sequences can beincluded in the same cassette (FIG. 1B, 1D). In this case a nucleotideloop, preferably containing 9 nucleotides, is inserted between the senseand antisense siRNA sequences. The resulting single PCR product includesthe U6 promoter, the siRNA sense and antisense encoding sequences in theform of a stem-loop, the Pol III terminator sequence, and the “stuffer”tag sequence (FIG. 1D). To construct this cassette two 3′ primers areused. The first PCR reaction employs the 5′ U6 universal primer and a 3′primer complementary to 20 nucleotides of the U6 promoter, followed bysequences complementary to the siRNA sense encoding sequence and the 9nt. loop (FIG. 1B). One microliter of this first reaction is reamplifiedin a second PCR reaction that employs the same 5′ U6 primer and a 3′primer harboring sequences complementary to the 9 nt. loop appended tothe antisense strand, Ter and “stuffer” tag sequence (FIG. 1B).

In another embodiment, a one step PCR reaction is conducted with asingle 3′ primer that harbors the sense, loop, antisense, Ter and“stuffer’ tag sequences (FIG. 1C). Although generally effective, thisapproach employs a considerably long and structured 3′ PCR primer thatwith some sequences may cause difficulties in obtaining the desired fulllength, double stranded PCR products.

PCR conditions are relatively standard for all siRNA genes since theregions complementary to the U6 promoter do not change. For theconstruction of several cassettes, optimal amplification was achieved ineach case using 1 minute for each PCR step and 55° C. as annealingtemperature. For direct transfections and testing of the PCR amplifiedsiRNA genes, the 5′ termini of the PCR primers may be modified, forexample, by phosphorylation, preferably using a DNA polynucleotidekinase and non-radioactive ATP. This modification of the primersstabilizes the PCR products intracellularly, thereby enhancing theefficacy of the PCR products.

Once the PCR reaction is completed, the products can be column purifiedfrom the primers, e.g., via a gel filtration column or by excising themdirectly from a gel following electrophoresis. The purified products canbe applied to cells following cationic liposome encapsidation and/orstandard transfection procedures, such as those described below and inco-pending Application Ser. No. 60/356,127, filed on Feb. 14, 2002,which is incorporated herein by reference. Intracellular expression ofthe transfected PCR products was detected by Northern blotting analyses(FIG. 3A), thus demonstrating good transfection efficiency.

Rapid Screening of Functional siRNAs and Accessible Target Sites UsingsiRNA-Encoding PCR Products.

An HIV rev sequence fused to the enhanced green fluorescent protein(EGFP)-coding sequence (Lee et al., 2002) was used to test the PCRamplified siRNA encoding DNA for efficacy in cells. This construct wasinserted in the Ecdysone-inducible pIND vector system (Invitrogen). Thevector was then transfected into 293 cells, which stably express thetrans-activator for the inducible promoter. Use of this system resultsin strong EGFP expression following addition of Ponasterone A(Invitrogen) to the culture media (FIG. 3A).

A stable cell line expressing both the trans-activator and targetconstructs may be preferable when multiple siRNA genes are being tested,but co-transfection with the target-EGFP fusion construct provides arapid and sensitive test for siRNA efficacy. Target sequence cDNAs canbe readily cloned into this inducible vector system to create thedesired EGFP fusion. Utilizing this system, an effective siRNA expressedfrom the PCR product will inhibit EGFP expression, allowing either FACSor microscopic based analyses of siRNA function.

To test the PCR approach, U6 cassettes containing either sense orantisense siRNA genes (FIG. 1A) or a hairpin construct encoding both thesense and antisense si-RNAs (FIG. 1C) were amplified. The PCR productswere column purified. The purified PCR products were then co-transfectedwith the inducible rev-EGFP fusion construct into the Ecdysonetransactivator expressing cell line. 48 hours post transfection,Ponasterone A was added to the culture to induce target mRNA expression.Using this system a strong and specific down regulation of EGFPexpression by the siRNAs was detectable 12 hours post induction (FIG.2). Transfection of a control cassette, such as a U6 expression cassetteexpressing only the sense (FIG. 2B), the antisense (FIG. 2C) or anirrelevant siRNA (not shown), had no effect on expression of EFGP.However, when cassettes expressing the sense and antisense siRNAs wereco-transfected with the target, or when a single cassette containing thehairpin siRNA gene was used, a specific and effective down regulation ofthe target was detected (FIGS. 2D and E). The best and most reproducibleinhibition (nearly 90%) was obtained with the hairpin siRNA expressingcassette. These results were reproduced independently 5 times. Theselected length and sequence of the 9 base loop (UUUGUGUAG) used forthese experiments is based upon phylogenetic comparisons of loops foundin several micro-RNA precursors. When using the above loop, the sequenceof the siRNA sense strand preferably should not include a U as the 3′base since this would create a stretch of 4 Uridines, which can serve asa Pol III terminator element.

The above results indicate that the transfection-PCR methodology of thepresent invention can be easily used to rapidly test siRNA targeting andfunction in cells.

An important element in the design of effective siRNAs is the selectionof siRNA/target sequence combinations that yield the best inhibitoryactivity. This can be accomplished using siRNAs and transfectionprocedures, but this can be costly and time consuming. By utilizing thePCR strategy, several siRNA genes can be simultaneously tested in asingle transfection experiment.

In order to facilitate the identification of functional siRNA genes, a“stuffer” tag sequence was inserted directly after the Pol IIItranscription terminator (see FIG. 1). By utilizing this tag, atransfected PCR cassette can be amplified from transfected cells and thesiRNA sequence subsequently identified (FIG. 1D). This can beaccomplished by utilizing the 5′ U6 universal primer and a primercomplementary to the tag sequence (FIG. 1D). The tag sequence can startwith the 6 Ts of the Ter sequence followed by a restriction site thatcan be used for subsequent cloning, and a “stuffer” of 6 extranucleotides (for a total of 18 nt). A mix of several siRNAs can besimultaneously co-transfected with the inducible target-EGFP cassetteinto the cell line containing the trans-activator. Twelve hours afteradding Ponasterone A, the EGFP negative and EGFP positive cells can becollected by FACS sorting, and the DNAs harvested from both fractions.The isolated PCR products can then be transfected for a second round ofselection and amplification to select those siDNA genes that express themost potent siRNAs. The resultant PCR products can then be cloned andsequenced. The functional siRNA can be identified since it would beabsent in the cells still expressing EGFP but present in the EGFPnegative fraction.

Using variations of the above approach, several expression cassettes maybe created and used to simultaneously screen for siRNA sensitive targetsites on any given mRNA. The target sequence may be fused to EFGP or asimilar reporter, and screening can be rapidly accomplished via FACSanalyses and sorting. This strategy can be utilized for endogenoustargets when there is a positive selection or a FACS sortable phenotypeavailable. An amplification strategy in accordance with the presentinvention offers a rapid and inexpensive approach for intracellularexpression of siRNAs and subsequent testing of target site sensitivityto down-regulation by siRNAs.

The present invention is further detailed in the following Examples,which are offered by way of illustration and are not intended to limitthe invention in any manner.

Example 1 Target Construction and Location of the siRNA Target Site

The HIV-rev sequence followed by the EGFP gene cloned in thepIND-inducible vector (Invitrogen) as previously described (Lee et al.,2002) was selected as a target site for siRNA. The selection of theaccessible target site for the siRNA was based on previous work and wasshown to be an effective siRNA target using the U6 expression system(Lee et al., 2002). The sequence of the target site is: 5′GCCTGTGCCTCTTCAGCTACC 3′ [SEQ ID NO: 1], which is located 213nucleotides downstream of the rev-AUG start codon.

Example 2 Polymerase Chain Reaction

PCR reactions were performed using a plasmid containing the human U6promoter as template. The 5′ oligonucleotide (5′U6 universal primer) iscomplementary to 29 nucleotides at the 5′ end of the U6 promoter (bolditalics) 5′ATCGCAGATCTGGATCCAAGGTCGGGCAGGAAGAGGGCCT3′ [SEQ ID NO: 2] andwas used for all PCR steps. The 3′ oligonucleotides, which containeither the sense, antisense, or both sense and antisense, are depictedin FIG. 1 and described herein. The last 20 nucleotides at the 3′ end ofall 3′ PCR primers are complementary to the last 20 nucleotides of theU6 promoter which is: 5′GTGGAAAGGACGAAACACCG3′ [SEQ ID NO: 3]. All PCRreactions were carried out as follows: 1 min. at 94° C., 1 min at 55° C.and 1 min at 72° C. for 30 cycles. The PCR primers were kinased withnon-radioactive ATP prior to amplification and purified on Qiagencolumns prior to using them in the PCR reactions. The PCR products werealso purified on Quiagen columns.

The sequences of the siDNA encoding oligos are:

1. Sense for siRNA rev-5′CGAAAAGGCCTAAAAAAGGTAGCTGAAGAGGCACAGGCGGTGTTTCGTCCTTTCCACAAGATATATAA 3′[SEQ ID NO: 4] 2. Antisense for siRNA rev-5′CGAAAAGGCCTAAAAAAGCCTGTGCCTCTTCAGCTACCGGTGTTTCGTCCTTTCCACAAGATATATAA 3′[SEQ ID NO: 5] 3. Hairpin siRNA oligo 1- sense5′TACACAAAGGTAGCTGAAGAGGCACAGGCGGTGTTTCGTCCTTTCCACAAGATATATAA 3′[SEQ ID NO: 6] 4. Hairpin siRNA oligo 2-antisense5′CGAAAAGGCCTAAAAAAGCCTGTGCCTCTTCAGCTACCCTACACAAAGG 3′ [SEQ ID NO: 7]

The italicized sequences are the siRNA encoding sequences.

Example 3 Cell Lines and Culture Conditions

293 cells were grown DMEM (Irvine Scientific, Santa Ana, Calif.)supplemented with 10% fetal calf serum (Irvine Scientific), 1 mML-glutamine, and 100 units/ml of penicillin/streptomycin. TheEcdysone-inducible stable A293 clone has been previously described (Leeet al., 2002) and it was maintained in DMEM containing 100 μg/ml ofZeocin (Invitrogen).

Example 4 Transfection Conditions for siRNA-PCR Products

250 ng of the target plasmid were co-transfected with either: 1) 50 ngof the PCR cassette expressing the sense, and/or 50 ng of the cassetteexpressing the antisense siRNA; or 2) 100 ng of the single cassetteexpressing both the sense and antisense linked by a 9 nt. loop. As fewas 25 ng of the stem loop siRNA was effective in blocking targetexpression. An irrelevant stem-loop siRNA was used as an additionalcontrol and did not result in any effect on target expression (notshown).

To facilitate the transfection of the small amounts of PCR amplifiedDNA, 400 ng of Bluescript plasmid were added to each reaction to serveas carrier. 5 μM Ponasterone A was added to the culture media 48 hoursafter transfection, and the cells were analyzed for EGFP expression 12hours following induction. Transfections were performed in 6 well platesusing Lipofectamine Plus™ reagent (Life Technologies, GibcoBRL) asdescribed by the manufacturer. For microscope imaging, cells were grownand transfected on glass coverslips treated with 0.5% gelatin (Sigma).12 hours post-induction the coverslips were lifted from the 6 well plateand treated for 10 min. at room temperature with 4% PFA for cellfixation. Cell nuclei were visualized adding DAPI to the mountingsolution. Down regulation of the rev-EGFP mRNA was quantitated by FACSanalyses.

Example 5 Northern Analyses

Total RNA was isolated using RNA STAT-60 (TEL-TEST B Inc., Friendswood,Tex.) according to the manufacturer's instructions. 5 μg of total RNAwas fractionated by 8M-6% PAGE, and transferred onto Hybond-N+ membrane(Amersham Pharmacia Biotech). A ³²P-radiolabeled 21-mer probecomplementary to the si-antisense RNA was used for the hybridizationreactions, which were performed for 16 hours at 37° C. A 21-mer DNAoligonucleotide was electrophoresed alongside the RNA samples and usedas size and hybridization control (not shown).

Example 6 Direct Amplification of siRNAs from Cell Lysates

EGFP-negative and -positive cell fractions were collected by FACSsorting. The cell pellets were recovered immediately by centrifugationof the sorted fractions. The pellets were lysed in 50 mM KCl, 10mMTris-HCl (pH 8.0), 1.25 mM MgCl₂, 0.45% NP40, 0.45% Tween, and 0.75μg/μl Proteinase K at 37° C. overnight. After 10 minutes heatinactivation at 95° C., 3 μl of the cell lysates were used directly inPCR reactions.

Example 7

15 ng of the PCR amplified gene encoding the siRNA hairpin targeting theHIV-rev site, along with 15 ng of an irrelevant siRNA PCR product wereco-transfected with the inducible target-EGFP cassette into the 293 cellline expressing the trans-activator. Twelve hours post transfection,Ponasterone A was added to induce EGFP expression and the EGFP negativeand positive cells were FACS sorted. The cell pellets from both the EGFPnegative and positive cells were collected by centrifugation, lysedovernight in lysis buffer and the DNAs amplified directly by PCRutilizing the appropriate primer sets. Two different 3′ primers thatdiscriminate between the two different siRNA encoding DNA cassettes wereused. It was expected that the non-functional siRNA expression cassetteshould be detectable by PCR amplification in both cell fractions,whereas the functional siRNA expression cassette would only bedetectable in the EFGP negative fraction since its products would havefunctionally downregulated EGFP expression. The results of twoindependent experiments are shown in FIGS. 3B and C. In both cases, thenon-functional siRNA encoding gene was PCR amplified from all fractions(FIG. 3B), whereas the functional siRNA encoding expression constructwas primarily detected in the EGFP negative cell fractions (FIG. 3B).

Example 8

Anti-HIV U6+1 short hairpin siRNA (shRNA) PCR products were produced byPCR using a U6+1 promoter construct as template (pTZU6+1), a universal5′ primer, and a specific 3′ primer. The primers are shown in Table 1 inthe standard 5′ to 3′ orientation. The shRNAs were designed to betranscribed in the sense target sequence-loop-antisense targetsequence-UUUUU (pol3 terminator) format. The sequence of the universal5′ primer, which anneals to the 5′ end of the U6+1 promoter, also isshown in Table 1. Table 1 further shows the corresponding sequence ofthe 3′ end of expected PCR product (the upper, coding strand is shown inthe standard 5′ to 3′ orientation), beginning with the 3′ end of theU6+1 promoter, ending with the +1 start site of transcription, followedby the sequences coding for the hairpin RNA (sensetarget/loop/anti-sense), the Pol III terminator, Bgl2 site and extranucleotides. The sequences of the 3′ primers also are shown in Table 1following the sequence of each PCR product.

Table 1 also shows a SELEX 2144 tRNA^(Lys3)-tat/rev target 21-stemshRNA.

Example 9

PCR-amplified expression cassettes expressing anti-tat siRNA were foundto potently inhibit HIV infection. PCR amplified short hairpin RNAencoding genes U6+1NLS1(tat/rev)shRNA, versus the same gene in a plasmidvector (pBS U6+1 NLS1 (tat/rev)shRNA), in the amounts indicated in FIG.4, were co-transfected with 0.5 micrograms of HIV pNL4-3 into 293 cellsand the viral encoded p24 antigen output was measured over three days.As controls, empty vector backbones (pBS or pTZU6+1) or a triplemutation at sites 9, 10 and 11 of the anti-tat shRNA (U6+1mNLS1shRNA(PCR product) or pBSU6+1mNLS1shRNA (plasmid based system)) weretransfected as a PCR amplified gene with HIV-1. The results in FIG. 4show the several logs worth of inhibition obtained using cassettesproduced according to the present invention as compared to the same genein a plasmid vector.

FIG. 4 shows that the U6+1 tat/rev shRNA specifically mediatescomparable HIV inhibition at equivalent molar ratios, regardless ofwhether the cassette is transfected as a PCR product directly or is partof a plasmid backbone. This panel also illustrates that small amounts ofan shRNA construct can mediate substantial HIV inhibition withsusceptible target sites.

Example 10

FIG. 5 shows the persistence of HIV inhibition by shRNAs expressed fromPCR products. HIV co-transfection inhibition assays were performed asdescribed in Example 9, using the U6+1 tat/rev shoRNA U6+1 tat/revmutant shRNA constructs as positive and negative controls. Aftercollecting viral supernatant on day 3, the confluent 293 cells werereseeded in fresh medium at 10% confluency and allowed to expand foranother 3 days before collecting viral supernatants for assay. FIG. 5shows that PCR product-mediated HIV inhibition persists for at least sixdays under these experimental conditions.

Example 11

FIG. 6 shows the results of an HIV inhibition test using cloned U6+1 envshRNA constructs (Table 1). Each well of a 6-cluster plate containing293 cells at ˜50% confluency was co-transfected with 0.5 μg of clonedplasmid shRNA and 0.5 μg pNL4-3 proviral DNA per well of a 6-clusterplate using Lipfectamine Plus according to the manufacturer'sinstructions. Aliquots of viral supernatants were taken at the indicatedtimes and assayed for p24 antigen. Tat/rev, positive control forinhibition; Mtat/rev, negative control for inhibition mismatched withtarget site at positions 10, 11, and 12 relative to 5′ end of processedantisense strand.

Example 12

FIG. 7 shows the results of HIV inhibition assays, using 200 ng of eachU6+1 env shRNA PCR product purified from a set of PCR reactions. pTZU6+1is a negative control plasmid containing the U6+1 promoter.

Example 13

FIG. 8 shows HIV inhibition by U6+1 env shRNAs, using 100 ng each U6+1env shRNA PCR product purified from another set of PCR reactions. U6+1tat/rev shRNA and U6+1 tat/rev Mutant shRNA PCR products were includedas controls.

The publications and other materials used herein to illuminate thebackground of the invention, and provide additional details respectingthe practice of the invention, are incorporated herein by reference asif each was individually incorporated herein by reference.

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that the disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

TABLE 1 Universal 5′ primer

HIV-1 env 1 Construct

HIV-1 env 2 Construct

HIV-1 env 4 Construct

HIV-1 env 6 Construct

HIV-1 env 7 Construct

HIV-1 tat/rev: positive control

HIV-1 tat/rev: negative control (nucleotides in red are mismatched topNL4-3 sequence)

HIV-1 tat/rev: positive control with BsrG1 loop

HIV-1 nef target shRNA

HIV-1 pol target shRNA

HIV-1 rev target shRNA (site II)

SELEX 2144 tRNA^(Lys3)-tat/rev target 21-stem shRNA)

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1. An amplification-based method for producing at least one mammalianpromoter-containing expression cassette, comprising: (i) adding a doublestranded nucleic acid comprising a mammalian promoter to anamplification reaction mixture, wherein the double stranded nucleic acidhas a sense strand and an antisense strand and wherein each of the sensestrand and antisense strand has a 5′ end and a 3′ end, wherein themammalian promoter is capable of transcribing an RNA molecule inmammalian cells; (ii) adding a first oligonucleotide primer to thereaction mixture, wherein the first oligonucleotide primer iscomplementary to the 3′ end of the antisense strand of the doublestranded nucleic acid; (iii) adding a second oligonucleotide primer tothe reaction mixture, wherein the second oligonucleotide primer iscomplementary to the 3′ end of the sense strand of the double strandednucleic acid and wherein the second oligonucleotide primer comprises anucleotide sequence that is complementary to a nucleotide sequence thatencodes (1) a sense sequence of a double stranded siRNA molecule and (2)a terminator sequence; and (iv) amplifying the double stranded nucleicacid in a polymerase chain reaction amplification comprising (a)annealing the primers to the complementary strands of the doublestranded nucleic acid, (b) extending the annealed primers to produceextension products, (c) denaturing the extension products and (d)repeating the polymerase chain reaction amplification steps a sufficientnumber of times to produce a first amplified product comprising a firstmammalian promoter-containing expression cassette, wherein the firstmammalian promoter-containing expression cassette comprises (1) themammalian promoter, (2) the sense strand of the double stranded siRNAmolecule and (3) the terminator sequence.
 2. The method of claim 1,wherein the amplification product is purified.
 3. The method of claim 1,further comprising cloning the amplified product comprising themammalian promoter-containing siRNA expression cassette into a cloningvector.
 4. The method of claim 1, further comprising a second polymerasechain reaction amplification comprising: (a) adding a double strandednucleic acid comprising a mammalian promoter to an amplificationreaction mixture, wherein the double stranded nucleic acid has a sensestrand and an antisense strand and wherein each of the sense strand andantisense strand has a 5′ end and a 3′ end, wherein the mammalianpromoter is capable of transcribing an RNA molecule in mammalian cells;(b) adding a first oligonucleotide primer to the reaction mixture,wherein the first oligonucleotide primer is complementary to the 3′ endof the antisense strand of the double stranded nucleic acid; (c) addinga second oligonucleotide primer to the reaction mixture, wherein thesecond oligonucleotide primer is complementary to the 3′ end of thesense strand of the double stranded nucleic acid and wherein the secondoligonucleotide primer comprises a nucleotide sequence that iscomplementary to a nucleotide sequence that encodes (1) an antisensesequence of the double stranded siRNA molecule and (2) a terminatorsequence; and (d) amplifying the double stranded nucleic acid in apolymerase chain reaction amplification comprising (i) annealing theprimers to the complementary strands of the double stranded nucleicacid, (ii) extending the annealed primers to produce extension products,(iii) denaturing the extension products and (iv) repeating thepolymerase chain reaction amplification steps a sufficient number oftimes to produce a second amplified product comprising a secondmammalian promoter-containing expression cassette, wherein the secondmammalian promoter-containing expression cassette comprises (1) themammalian promoter, (2) the antisense strand of the double strandedsiRNA molecule and (3) the terminator sequence.
 5. The method of claim4, wherein the first and second amplification products are purified. 6.The method of claim 4, further comprising cloning the first amplifiedproduct comprising the first mammalian promoter-containing expressioncassette into a cloning vector and cloning the second amplified productcomprising the second mammalian promoter-containing expression cassetteinto a cloning vector.
 7. The method of claim 1, wherein the mammalianpromoter is a Pol III promoter.
 8. The method of claim 7, wherein thePol III promoter is a mammalian U6 promoter.
 9. The method of claim 8,wherein the U6 promoter is a human U6 promoter.
 10. The method of claim1, wherein the sequence encoding the terminator sequence comprises asequence of about 4-6 deoxyadenosines.
 11. The method of claim 10,wherein the sequence encoding the terminator sequence comprises asequence of 6 deoxyadenosines.
 12. The method of claim 1, wherein thesecond oligonucleotide primer further comprises a tag sequence toidentify functional siRNA encoding sequences.
 13. The method of claim12, wherein the tag sequence further comprises a restriction site usefulfor cloning.
 14. The method of claim 4, further comprising the step oftransfecting a mammalian cell in vitro with the first amplifiedmammalian promoter-containing expression cassette and with the secondamplified mammalian promoter-containing expression cassette, wherein thesense strand of the double stranded siRNA molecule and the antisensestrand of the double stranded siRNA molecule are expressed in thetransfected cell, whereby a double stranded siRNA molecule is producedin the transfected cell.
 15. The method of claim 14, wherein one or moreof the oligonucleotide primers are modified.
 16. The method of claim 15,wherein one or more of the oligonucleotide primers are modified byphosphorylation.
 17. The method of claim 14, further comprising the stepof screening for a target site on mRNA sensitive to the double strandedsiRNA molecule produced.
 18. An amplification-based method for producinga mammalian promoter-containing expression cassette, comprising: (i)adding a double stranded nucleic acid comprising a mammalian promoter toan amplification reaction mixture, wherein the double stranded nucleicacid has a sense strand and an antisense strand and wherein each of thesense strand and antisense strand has a 5′ end and a 3′ end, wherein themammalian promoter is capable of transcribing an RNA molecule inmammalian cells; (ii) adding a first oligonucleotide primer to thereaction mixture, wherein the first oligonucleotide primer iscomplementary to the 3′ end of the antisense strand of the doublestranded nucleic acid; (iii) adding a second oligonucleotide primer tothe reaction mixture, wherein the second oligonucleotide primer iscomplementary to the 3′ end of the sense strand of the double strandednucleic acid and wherein the second oligonucleotide primer comprises anucleotide sequence that is complementary to a nucleotide sequence thatencodes (1) an antisense sequence of a double stranded siRNA moleculeand (2) a terminator sequence; and (iv) amplifying the double strandednucleic acid in a polymerase chain reaction amplification comprising (a)annealing the primers to the complementary strands of the doublestranded nucleic acid, (b) extending the annealed primers to produceextension products, (c) denaturing the extension products and (d)repeating the polymerase chain reaction amplification steps a sufficientnumber of times to produce an amplified product comprising a firstmammalian promoter-containing expression cassette, wherein the firstmammalian promoter-containing expression cassette comprises (1) themammalian promoter, (2) the antisense strand of the double strandedsiRNA molecule and (3) the terminator sequence.
 19. The method of claim18, further comprising a second polymerase chain reaction amplificationcomprising: (a) adding a double stranded nucleic acid comprising amammalian promoter to an amplification reaction mixture, wherein thedouble stranded nucleic acid has a sense strand and an antisense strandand wherein each of the sense strand and antisense strand has a 5′ endand a 3′ end, wherein the mammalian promoter is capable of transcribingan RNA molecule in mammalian cells; (b) adding a first oligonucleotideprimer to the reaction mixture, wherein the first oligonucleotide primeris complementary to the 3′ end of the antisense strand of the doublestranded nucleic acid; (c) adding a second oligonucleotide primer to thereaction mixture, wherein the second oligonucleotide primer iscomplementary to the 3′ end of the sense strand of the double strandednucleic acid and wherein the second oligonucleotide primer comprises anucleotide sequence that is complementary to a nucleotide sequence thatencodes (1) an sense sequence of the double stranded siRNA molecule and(2) a terminator sequence; and (d) amplifying the double strandednucleic acid in a polymerase chain reaction amplification comprising (i)annealing the primers to the complementary strands of the doublestranded nucleic acid, (ii) extending the annealed primers to produceextension products, (iii) denaturing the extension products and (iv)repeating the polymerase chain reaction amplification steps a sufficientnumber of times to produce a second amplified product comprising asecond mammalian promoter-containing expression cassette, wherein thesecond mammalian promoter-containing expression cassette comprises (1)the mammalian promoter, (2) the sense strand of the double strandedsiRNA molecule and (3) the terminator sequence.
 20. The method of claim19, further comprising the step of transfecting a mammalian cell invitro with the first amplified mammalian promoter-containing expressioncassette and with the second amplified mammalian promoter-containingexpression cassette, wherein the sense strand of the double strandedsiRNA molecule and the antisense strand of the double stranded siRNAmolecule are expressed in the transfected cell, whereby a doublestranded siRNA molecule is produced in the transfected cell.